The administration of soluble protein antigens via the oral route has been described as a means of inducing sytemic immunological tolerance (oral tolerance). We had previously explored the mechanisms of oral tolerance induction with the use of ovalbumin (OVA) T cell receptor (TCR)-transgenic mice, and demonstrated the importance of IL-12 for the regulation of two mechanism of oral tolerance, clonal anergy/deletion, and the generation of TGFb-producing T cells. Thus, it was demonstrated that the administration of anti-IL-12 to intact OVA TCR-transgenic mice fed OVA, enhanced both TGFb production and apoptosis of antigen-specific T cells. To further understand the effect of anti-IL-12 treatment on oral tolerance, we conducted a series of studies on the role of various cytokines in TGF-b production, both in vivo and in vitro. We demonstrated that the systemic administration of antibodies to IL-12 to ovalbumin (OVA)-T cell receptor (TCR) transgenic mice fed high doses of OVA, followed by systemic OVA challenge, substantially enhances TGF-b production after re-stimulation of peripheral T cells in vitro. Furthermore, we showed that naVve (CD4+/MEL-14hi) OVA-TCR-T cells stimulated with OVA-pulsed dendritic cells in vitro produce 4-5 fold higher amounts of TGF-b when cultured with anti-IL-12 or anti-IFN-g. IL-4 was not required for TGF-b production, however it appeared to indirectly enhance TGF-b production by promoting the growth of TGF-b producing cells. Taken together, our findings demonstrate that IL-12 and IFN-g are important negative regulators of TGF-b production both in vivo and in vitro. They thus explain the ability of anti-IL-12 treatment to enhance oral tolerance as discussed above. In further studies carried out with Dr. Robert Seder (MIS/LCI/NIAID), the production of TGFb by T cells following primary and secondary stimulation in vitro was studied using T cells from TCR-transgenic, as well as IL-4 and IFN-g knockout mice. It was determined that conditions favoring either high IL-4 production (e.g., the presence of IL-4 in the priming cultures), or low IFN-g production (e.g., culture with anti-IL-12 and anti-IFN-g) resulted in the emergence of TGFb-producing T cells after a secondary in vitro stimulation. However, it was also determined that IL-4 was not absolutely required for TGFb -production, consistent with our prior findings.